Method for alleviating gastric mucosal bleeding

ABSTRACT

Gastric mucosal bleeding can be prevented or substantially diminished by parenterally administering a therapeutically effective amount of glucagon to a subject experiencing such bleeding.

United States Patent [1 1 Lin [ Dec. 16, 1975 METHOD FOR ALLEVIATINGGASTRIC MUCOSAL BLEEDING [75] Inventor: Tsung-Min Lin, Indianapolis,Ind.

[73] Assignee: Eli Lilly and Company, Indianapolis,

Ind.

[22] Filed: June 7, 1974 [21] Appl. No.: 477,213

[52] US. Cl. 424/177 51 Int. c1. A61K 37/00 58 Field of Search 424/177,178; 260/1125, 260/1127 [56] References Cited UNITED STATES PATENTS3,522,229 7/1970 Yamamoto et al. 424/177 OTHER PUBLICATIONS Lin et al.,Chem. Abstr. 75: 148,178h (1971).

Primary Examine r-Lewis Gotts Assistant Examiner-Reginald J. SuyatAttorney, Agent, or Firm--William C. Martens, Jr.; Everet F. Smith [57]ABSTRACT Gastric mucosa] bleeding can be prevented or substantiallydiminished by parenterally administering a therapeutically effectiveamount of glucagon to a subject experiencing such bleeding.

6 Claims, 6 Drawing Figures U.S. Patent Dec. 16, 1975 CATIONCONCENTRATION IN RECOVER INSTILLATE (meg/l) Sheet 6 of 6 3,927,212

AMINOPYRINE 5 mg/kg-hr ASPIRIN 5mg/ml Y Glucugon T 50pg/ kg s.c.

oo Aspirin o-----0 Aspirin-Glucugon 30 MINUTE PERIODS FIG. 6

METHOD FOR ALLEVIATING GASTRIC MUCOSAL BLEEDING BACKGROUND OF THEINVENTION It is well documented that carboxylic acids, such as acetic,propionic, butyric, and, most significantly, acetylsalicyclic acid(aspirin), in a highly acidic pH gastric environment, can diffusethrough the gastric mucosa, producing injury to the mucosal barrier.Once the integrity of the mucosal barrier has been damaged, backdiffusion of acid results, and this is accompanied by gastric bleeding.

It has now been discovered that gastric bleeding can be alleviated byadministering glucagon to a subject experiencing such bleeding.Furthermore, gastric bleeding can be prevented or substantiallydiminished by administering glucagon to a subject who can be reasonablyexpected to experience gastric bleeding.

structurally, glucagon is a single polypeptide chain of 29 amino acids.The amino acid sequence of glucagon was established by Bromer et al., J.Am. Chem. 800., 79, 2807 (1957). Glucagon exhibits a hyperglycemiceffect and, thus, has long been recognized as useful in treatinghypoglycemia. However, it has never been considered that glucagon wouldbe active in alleviating gastric bleeding. It is to the use of glucagonin treatment of gastric bleeding that this invention is directed.

DETAILED DESCRIPTION OF THE INVENTION Broadly, this invention isdirected to a method for alleviating gastric mucosal bleeding whichcomprises parenterally administering to a mammal a therapeuticallyefl'ective amount of glucagon.

Therefore, the core of this invention resides in the discovery thatglucagon is effective in alleviating, terminating, or preventing gastricbleeding. The glucagon is administered to a warm-blooded animal who isexperiencing gastric bleeding, or it is administered to one who mightreasonably be expected to experience gastric bleeding.

The glucagon dosage level is an amount sufficient to diminish or toterminate gastric bleeding. Generally, the dosage level will be withinthe range of from about 1.0 pg. to about 200 pg. per kg. body weight ofthe recipient, and, preferably, from about 25 pg. to about 150 pg. perkg. body weight, and, most preferably, from about 25 pg. to about 100pg. per kg. body weight. Furthermore, thetotal dosage can be single orcumulative. By cumulative is meant a dosage measured in terms ofmultiple individual smaller doses, with each such individual dose beingadministered prior to completion of the recipients response to theprevious dose.

Any of the generally employed methods for administering glucagon can beemployed. These include all of the usual parenteral routes, for example,intravenous, intraperitoneal, subcutaneous and intramuscular.Preferably, however, the glucagon is administered subcutaneously (s.c.).

Glucagon is active in preventing or retarding gastric bleeding whenadministered to a mammal which is susceptible to such malady or inalleviating or terminating gastric bleeding when administered to onewhich exhibits such bleeding. The cause of the gastric bleeding is notcrucial to the effectiveness of the glucagon in achieving alleviation.Preferably, however, this inven- 2 tion is directed to treatment ofgastric bleeding when such bleeding has been initiated or is reasonablyexpected to occur from ingestion of an ulcerogenic antiinflammatoryagent. Typical such agents include ace- 5 tylsalicyclic acid (aspirin),indomethacin, phenylbutazone, and the like. Most typically, the gastricbleeding will result from ingestion of acetylsalicyclic acid.

The recipient of the glucagon can be any mammal, human or non-human, whois experiencing or can reasonably be expected to experience gastricbleeding.

The alleviation or prevention of gastric mucosal bleeding byadministration of glucagon is illustrated by the following experimentalprocedure and data. The particular procedure, however, is not intendedto be limiting upon the broad scope of the discovery of this invention.

EXAMPLE Four female dogs, weighing 22-25 kg. each, were surgicallyaltered to provide a vagally innervated gastric fistula as well as adenervated fundic (Heidenhain) pouch. The dogs were so prepared at leastone month prior to any use experimentally. No dog was usedexperimentally more than once per each 14 day period.

The experimental procedure was commenced by fasting each dog for 18hours, although each was permitted to have free access to water. Underthese basal conditions there was no measurable gastric acid secretion.The pouch then was washed throughly with 0.1 N HCl. Additional 0.1 NI-ICl (25 ml.) then was instilled (infused) into the pouch and wasmaintained there for 30 minutes. The pouch then was drained, and the HClwas saved for analysis. Immediately, 25 ml. of fresh 0.1 N HCl wasinstilled in the pouch and maintained there for a 30 minute period. Thisprocess was repeated each 30 minutes for a total of 6 hours. Thus, atotal of 12 portions (designated herein as Periods l-l2) of 0.1 N HClwere instilled into and recovered from the pouch. All instillates(infusates) except those of Periods 4, 5, and 6 were identical incomposition. To those of Periods 4, 5, and 6 were added 5 mg/ml. (27.8mmoles) of acetylsalicyclic acid (aspirin).

Blood flow to the gastric mucosa of each of the dogs was measured byclearance of plasma aminopyrine into the HCl instillate. Thus,aminopyrine was administered to each of the dogs. It was maintained at asteady level in the plasma of the dog by continuous infusionintravenously at a rate of 5 mg/kg.-hr. following an initial dose of 20mg./kg. administered prior to commencement of addition of HCl to theHeindenhain pouch. The presence and amount of aminopyrine in theinstillate was determined by extracting it from the instillate using aprocedure described by Jacobson et al., Blood Flow and Secretion in theStomach, Gastroenterology, 52, (1967) pp. 414-420; and Jacobson et al.,Gastric Secretion in Relation to Mucosal Blood Flow studied by 2Clearance Technic, Jour. Clin. Invest, 45, (1966) pp. 1-13, with theminor modification described in Lin et al., Effect of Glucagon onPentagastric-induced Gastric Acid Secretion and Mucosal Blood Flow inthe Dog, Gastroenterology, 61 (1971) pp. 328-331. Concentration ofaminopyrine in the extract was then determined by its absorption at 260mp measured spectrophotometrically.

Each of the four dogs also was administered Cr by slow infusionintravenously of Cr C1 (25 pCi/kg) over a 3 hour period on the day priorto testing. Plasma transferrin thereby was tagged with Cr, and this pro-3 vided a ready measure of plasma transferrin and, in turn, a'measure ofany loss of plasma from the blood to the lumen. The amount of Cr in theblood and the amount in the infusate were monitored using a'y-spectrometer, and any loss of plasma protein thereby was calculated.

The preceding description constitutes a definition of the controlsequence. All four dogs were treated as aforedescribed, and therefromcontrol data were generated. However, 14 days prior to carrying out thecontrol sequence, the same four dogs were treated as aforedescribed withthe exception that each additionally received glucagon. The glucagon wasadministered subcutaneously (s.c.) in two separate doses of 50 ug/kg.each. The doses were administered at the outset of Periods and 7, thatis, simultaneously with administration of the second infusate containingacetylsalicyclic acid and upon removal of the third (last) infusatecontaining acetylsalicyclic acid.

In addition to the aforementioned analytical techniques, the infusatewithdrawn from each of the four dogs after each of the 12 periods wassubjected to further analysis.

The extent of gastric bleeding was determined by measuring the amount ofhemoglobin present in the infusate relative to the hemoglobinconcentration in the blood of the respective dog. Hemoglobin wasmeasured as acid hematin in accordance with Cohen et al., TheColorimetric Determination of Hemoglobin, Jour. Biol, Chem, 39, (1919)pp. 489-496.

Acidity of the recovered infusate was determined by titration to pH 7with 0.05 N NaOH, and the concentration of the sodium (Na potassium(K*), calcium (Ca**), and magnesium (Mg ions were measured by atomicabsorption.

Results from the testing of the four dogs under these conditions aredepicted herein by means of the graphs provided.

FIG. 1 depicts the effect of aspirin and aspirin-glucagon on the volumeof recovered instillate from the Heidenhain pouch of the four dogs. Theabscissa represents the twelve periods of thirty minutes each, and theordinate presents the volume in milliliters of instillate recovered.

FIG. 2 depicts the effect of aspirin and aspirin-glucagon on thehydrogen ion (II concentration in the recovered instillate from theHeidenhain pouches of four dogs. Values represent the means isem. Theabscissa represents the 12 periods of minutes each, and the ordinatepresents the hydrogen ion concentration in milliequivalents (meq) perliter (1).

FIG. 3 depicts the effect of aspirin and aspirin-glucagon on theclearance of plasma aminopyrine from the Heidenhain pouches of the fourdogs. Values represent the means i s.e.m. The abscissa represents the 12periods of thirty minutes each, and the ordinate presents aminopyrineclearance in milliliters per minute.

FIG. 4 depicts the effect of aspirin and aspirin-glucagon on hemmorrhageinto the instillate of the Heidenhain pouches of the four dogs. Theabscissa represents the 12 periods of 30 minutes each, and the ordinatepresents total blood in milliliters.

FIG. 5 depicts the effect of aspirin and aspirin-glucagon on the loss ofplasma transferrin-Cr into the instillate of the Heidenhain pouches ofthe four dogs. Values represent the means s.e.m. The abscissa representsthe 12 periods of 30 minutes each, and the ordinate 4 presentsmicroliters ([Ll.) of plasma per milliliter (ml.) of instillate.

FIG. 6 depicts the effect of aspirin and aspirin-glucagon on the netefflux (concentration) of Na, K", Ca, and Mg in the instillate of theHeidenhain pouches of the four dogs. Values represent the means'z':s.e.mm. The abscissa represents the 12 periods of 30 minutes each, andthe ordinate presents the cation concentrations in milliequivalents perliter (meq/l) of recovered instillate.

As is apparent from FIG. 1, during the three control periods, 24.2 to 25ml. of acid instillate were collected from the pouch at 30 minuteintervals. There was no significant difference in volume of instillaterecovered during the control periods of the aspirin test when comparedwith those of the aspirin-glucagon test.

After the assault with a combination of 0.1 N HCl and aspirin for three30 minute periods, a gradual but significant increase in volume ofrecovered instillate resulted. This increase continued for at least 3hours after cessation of aspirin administration, and the peak increasewas 7.3 ml. above control. Administration of glucagon subcutaneously(s.c.) in two doses of 50 ug/kg, each and 60 minutes apart,significantly reduced the volume of instillate recovered during Periods6-8 when compared to the volume recovered by administration of aspirinalone. The volume reduction amounted to a total of 17.2 ml.

The hydrogen ion (I-I concentration in the recovered installate fromboth series of tests remained essentially steady at meq/l beforeadministration of aspirin (FIG. 2). A significant decrease of Hconcentration was seen 60 minutes after assault with HCl and aspirin.The reduction in acid concentration persisted for about three hoursafter termination of aspirin administration. The difference in acidconcentration between administration of aspirin alone and administrationof the glucagon-aspirin combination was not significant.

Gastric mucosal blood flow is indicated by clearance of plasmaaminopyrine (FIG. 3). Clearance of plasma aminopyrine was significantlyincreased by administration of aspirin, and this increase persisted forabout three hours after termination of aspirin administration. Theadministration of glucagon significantly reduced the clearance ofaminopyrine and, thus, of the mucosal blood flow in Periods 6-8. Theextent of reduction indicated a mucosal blood flow reduced to an amounteven below that obtained prior to aspirin administration.

Mucosal hemmorrhagingcaused by aspirin was significantly reducedthroughoutthe test by administration of glucagon (FIG. 4). The mean oftotal blood loss during a 4.5 hour period was 12.3 ml. in the aspirincontrols, whereas administration of glucagon reduced total blood loss toonly 6.8 ml.

The concentration of plasma protein in the lumen, calculated bymeasuring Cr transferrin in the instillate and in the plasma, was, inthe aspirin controls, 46.4 ul/ml. over a 4.5 hour period. Administrationof glucagon reduced this to 19.1 pJ/ml, a 58.8 percent reduction (FIG.5). There was a high correlation between loss of hemoglobin and loss ofplasma protein (r 0.96).

The net efflux of sodiumion (Na from mucosa to lumen provides asensitive index of the integrity of the gastric mucosal barrier. 30minutes after assault with aspirin and before there was any significantloss of hydrogen ion from lumen to mucosa, the Na concentration hadalready doubled in both the aspirin and the aspirin-glucagon series oftests (FIG. 6). The maximum increases in Na concentration occurredduring or immediately after assault with aspirin and were four times thevalues obtained before administration of aspirin. The mean Naconcentrations following injections of glucagon were not significantlydifferent from those obtained from aspirin administration alone.

Aspirin administration increased the net potassium ion (14*)concentration by a factor of 2.3 to 3.5 during the 90 minutes ofadministration. This increase in net K efflux persisted after withdrawalof aspirin from the test solution and was not significantly altered atany point in the test by administration of glucagon.

The concentrations of calcium ion (Ca and magnesium ion (Mg were bothsignificantly increased by aspirin administration. As shown by Periods4-7, the average concentration of Ca increased by a factor of 5.2 afteradministration of aspirin. Although the mean values for Ca were lower inthe glucagon-treated series than in that in which aspirin alone wasadministered, the differences of means were not statisticallysignificant.

The Mg concentrations was increased by a factor of 3.2 uponadministration of aspirin. The mean values for Mg concentration wereslightly lowered by glucagon administration; however, these differenceswere not statistically significant.

The results presented hereinabove agree with the recognized finding thataspirin in an acidic environment disrupts the integrity of the gastricmucosal barrier. The above results demonstrate that upon disruption ofthe integrity of the barrier, typical responses were noted, such asincrease in the net efflux of sodium and potassium ions, increase involume of recovered test solution, and loss of H from lumen to mucosa.There was sustained hemorrhage from mucosa to lumen after the assaultwith aspirin and for as long as the fundic mucosa was irrigated with 0.1N HCl.

The results further demonstrate that clearance of aminopyrine increasedsignificantly upon exposure of the Heidenhain pouch to aspirin.Theoretically, at least, the increased clearance of the aminopyrinecould be due in part to exudation of aminopyrine through injuredcapillaries. However, the increase in mucosal clearance of aminopyrinewas approximately 150 ml. per each 30 minutes (5 mL/min X 30 min., FIG.3), whereas the total loss of plasma to lumen was less than 2 ml. pereach 30 minutes (60 ,ul/ml X 30 min., FIG. 5). Therefore, the increasein aminopyrine clearance in fact represents a true increase in mucosalblood flow.

The administration of glucagon effected a significant reduction inclearance of aminopyrine (FIG. 3). The reduction in aminopyrineclearance caused by glucagon is indicative of a genuine reduction inmucosal blood flow.

Glucagon therefore significantly counteracted the action of aspirin withrespect to mucosal blood flow. As is evident from FIG. 4, glucagon alsocaused substantial reduction in mucosal hemorrhage. However, thisreduction does not appear to be directly dependent upon the reducedmucosal blood flow.

That this is true is evident from a comparison of the aminopyrineclearance (FIG. 3) with the total blood (FIG. 4). The reduction inmucosal blood flow (aminopyrine clearance) due to administration ofglucagon was evident from the outset of administration (Period 5) andthrough Periods 6 and 7. However, its effect had virtually expired bythe end of Period 8. In contradistinction, the total blood decreasedfrom the outset of glucagon administration (Period 5), and the reductioncontinued to be in evidence through to and beyond the end of the test(Period 12). Therefore, the reduced hemorrhaging was distinctively inexcess of that which would be attributable to the recognized reductionin mucosal blood flow.

It is apparent from the above, therefore, that glucagon has significantutility in arresting gastric bleeding in subjects experiencing same orin preventing or diminishing gastric bleeding in subjects susceptiblethereto, and caused, for example, by ingestion of an ulcerogenicanti-inflammatory agent, particularly aspirin, or by the presence of agastric ulcer.

I claim:

1. A method for alleviating gastric mucosal bleeding which comprisesparenterally administering to a mammal a therapeutically effectiveamount of glucagon.

2. Method of claim 1, in which the glucagon is administeredsubcutaneously.

3. Method of claim 1, in which a total of from about 1.0 .tg to about200 ug. of glucagon is administered per kilogram of body weight.

4. Method of claim 3, in which a total of from about 25 ug. to about 150ug. of glucagon is administered per kilogram of body weight.

5. Method of claim 4, in which a total of from about 25 82 g. to aboutug. of glucagon is administered per kilogram of body weight.

6. Method of claim 1, in which the total dosage of glucagon isadministered in multiple cumulative doses.

UNITED STATES PATENT AND TRADEMARK OFFICE CERTIFICATE OF CORRECTIONPATENT NO. 5,927,2 DATED December 16, 1975 |N\/}ENTOR(S) Tsung-Min LinIt is certified that error appears in the above-identified patent andthat said Letters Patent are hereby corrected as shown below:

Column t, line 6, "s.e.mm." should read --s.e.m.--.

Column 6, line #7, "25 82 g." should read Signed and Scaled thisTwenty-eighth Day of June 1977 [SEAL] Attest:

RUTH C. MASON Arresting Officer C. MARSHALL DANN Commissioner oj'larenlsand Trademarks

1. A METHOD FOR ALLEVIATING GASTRIC MUCOSAL BLEEDING WHICH COMPRISESPARENTERALLY ADMINISTERING TO A MAMMAL A THERAPEUTICALLY EFFECTIVEAMOUNT OF GLUCAGON.
 2. Method of claim 1, in which the glucagon isadministered subcutaneously.
 3. Method of claim 1, in which a total offrom about 1.0 Mu g to about 200 Mu g. of glucagon is administered perkilogram of body weight.
 4. Method of claim 3, in which a total of fromabout 25 Mu g. to about 150 Mu g. of glucagon is administered perkilogram of body weight.
 5. Method of claim 4, in which a total of fromabout 25 82 g. to about 100 Mu g. of glucagon is administered perkilogram of body weight.
 6. Method of claim 1, in which the total dosageof glucagon is administered in multiple cumulative doses.